Molecular identification of Borrelia genus in questing hard ticks from Portugal: Phylogenetic characterization of two novel Relapsing Fever-like Borrelia sp

In the last decades, several studies have reported pathogenic species of Borrelia related to those that cause Tick-borne Relapsing Fever (RF), but unexpectedly suggesting their transmission by hard ticks, known vectors of Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species, rather than by soft ticks. This study was designed to update the presence of B. burgdorferi s.l. species in ticks from several districts of mainland Portugal, where Ixodes ricinus had been previously described. Ticks (a total of 2915 specimens) were collected in seven districts throughout the country, and analyzed using molecular methods. Three nested-PCR protocols, targeting the flagellin gene (flaB), the intergenic spacer region (IGS) located between 5S and 23S rRNA, and the glpQ gene, and a conventional PCR targeting the 16S rRNA, were used for Borrelia DNA detection. Borrelia DNA was detected in 3% of the ticks from Braga, Vila Real, Lisboa, Setúbal, Évora and Faro districts. The obtained amplicons were sequenced and analyzed by BLASTn, and 15/63 (24%) matched with homologous sequences from Borrelia lusitaniae and 15/63 (24%) with B. garinii, being these the most prevalent species. DNA from B. burgdorferi sensu stricto (s.s.), B. valaisiana and B. afzelii were detected in 7/63 (11%), 6/63 (10%), and 2/63 (3%) of the specimens, respectively. Unexpectedly, DNA sequence (flaB) analysis from eight (13%) samples, two from Rhipicephalus sanguineus and six from Haemaphysalis punctata tick species, revealed high homology with RF-like Borrelia. Phylogenetic analyses obtained from three genetic markers (16S rRNA, flaB, and glpQ) confirmed their congruent inclusion in a strongly supported RF cluster, where they segregated in two subgroups which differ from the other Relapsing Fever species. Therefore, the results confirm the circulation of multiple species of B. burgdorferi s.l. over a wide geographic range, covering most of the Portuguese mainland territory. Surprisingly, the obtained data also revealed two putative Relapsing Fever-like Borrelia species in different species of hard ticks, possibly disclosing the circulation of novel RF-like Borrelia species with different associated tick vectors.